This project examines the relationship between protein structure and function by analyzing the roles of specific amino acid residues in the conversion of the inactive zymogen trypsinogen to its active counterpart trypsin. The activation of trypsinogen is a model for the activation of homologous- serine proteases involved in blood clotting, fibrinolysis and complement activation. This study will probe the forces governing protein stability and the mechanism of protein conformational change. This approach contrasts with typical mutational analyses of protein stability by studying the transition between two well characterized protein conformations, rather than the transition between denatured and native protein. The effects of specific amino acid substitutions on the structure and activity of trypsinogen and trypsin will be monitored using a variety of structural and functional probes. These techniques will include activity measurements, ligand binding, cd spectroscopy and nmr spectroscopy. The x-ray crystal structure of representative mutants will be solved. In addition, mutations will be identified which produce catalytically active trypsinogen. These experiments will define the structural features which are responsible for the high activity of the zymogens of such physiologically important serine proteases as tissue plasminogen activator and urokinase. Alternate strategies for stabilizing the active protease conformation are also important for the design of proteases with novel specificities, where progress has been limited by the instability of the S1 binding pocket.